Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont.
Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio.
Am J Physiol Heart Circ Physiol. 2021 May 1;320(5):H1822-H1835. doi: 10.1152/ajpheart.00673.2020. Epub 2021 Mar 5.
Cardiac myosin binding protein-C (cMyBP-C) is a thick filament protein that influences sarcomere stiffness and modulates cardiac contraction-relaxation through its phosphorylation. Phosphorylation of cMyBP-C and ablation of cMyBP-C have been shown to increase the rate of MgADP release in the acto-myosin cross-bridge cycle in the intact sarcomere. The influence of cMyBP-C on Pi-dependent myosin kinetics has not yet been examined. We investigated the effect of cMyBP-C, and its phosphorylation, on myosin kinetics in demembranated papillary muscle strips bearing the β-cardiac myosin isoform from nontransgenic and homozygous transgenic mice lacking cMyBP-C. We used quick stretch and stochastic length-perturbation analysis to characterize rates of myosin detachment and force development over 0-12 mM Pi and at maximal (pCa 4.8) and near-half maximal (pCa 5.75) Ca activation. Protein kinase A (PKA) treatment was applied to half the strips to probe the effect of cMyBP-C phosphorylation on Pi sensitivity of myosin kinetics. Increasing Pi increased myosin cross-bridge detachment rate similarly for muscles with and without cMyBP-C, although these rates were higher in muscle without cMyBP-C. Treating myocardial strips with PKA accelerated detachment rate when cMyBP-C was present over all Pi, but not when cMyBP-C was absent. The rate of force development increased with Pi in all muscles. However, Pi sensitivity of the rate force development was reduced when cMyBP-C was present versus absent, suggesting that cMyBP-C inhibits Pi-dependent reversal of the power stroke or stabilizes cross-bridge attachment to enhance the probability of completing the power stroke. These results support a functional role for cMyBP-C in slowing myosin detachment rate, possibly through a direct interaction with myosin or by altering strain-dependent myosin detachment via cMyBP-C-dependent stiffness of the thick filament and myofilament lattice. PKA treatment reduces the role for cMyBP-C to slow myosin detachment and thus effectively accelerates β-myosin detachment in the intact myofilament lattice. Length perturbation analysis was used to demonstrate that β-cardiac myosin characteristic rates of detachment and recruitment in the intact myofilament lattice are accelerated by Pi, phosphorylation of cMyBP-C, and the absence of cMyBP-C. The results suggest that cMyBP-C normally slows myosin detachment, including Pi-dependent detachment, and that this inhibition is released with phosphorylation or absence of cMyBP-C.
心肌肌球蛋白结合蛋白 C（cMyBP-C）是一种粗肌丝蛋白，通过磷酸化影响肌节刚度并调节心脏收缩-松弛。已经表明，cMyBP-C 的磷酸化和消融会增加完整肌节中肌球蛋白-肌动蛋白交联循环中 MgADP 的释放速率。cMyBP-C 对 Pi 依赖性肌球蛋白动力学的影响尚未得到研究。我们研究了 cMyBP-C 及其磷酸化对来自非转基因和纯合缺失 cMyBP-C 的转基因小鼠的去膜乳头肌条中 β-心脏肌球蛋白同工型的肌球蛋白动力学的影响。我们使用快速拉伸和随机长度扰动分析来表征在 0-12mM Pi 下以及在最大（pCa 4.8）和近半最大（pCa 5.75）Ca 激活下肌球蛋白分离和力发展的速率。用蛋白激酶 A（PKA）处理一半的肌条，以研究 cMyBP-C 磷酸化对肌球蛋白动力学对 Pi 敏感性的影响。增加 Pi 同样增加了有和没有 cMyBP-C 的肌肉中肌球蛋白交联桥的分离速率，尽管在没有 cMyBP-C 的肌肉中这些速率更高。用 PKA 处理心肌条时，当 cMyBP-C 存在于所有 Pi 时，分离速率加快，但当 cMyBP-C 不存在时则不会。在所有肌肉中，力发展的速率都随 Pi 增加而增加。然而，当存在 cMyBP-C 时，力发展的 Pi 敏感性降低，这表明 cMyBP-C 通过直接与肌球蛋白相互作用或通过改变依赖应变的肌球蛋白分离来抑制力冲程的反转，从而稳定交联桥的附着，从而提高完成力冲程的可能性。这些结果支持 cMyBP-C 在减慢肌球蛋白分离速率方面的功能作用，可能通过与肌球蛋白的直接相互作用或通过 cMyBP-C 依赖性粗丝和肌丝晶格的刚度改变应变依赖性肌球蛋白分离来实现。PKA 处理降低了 cMyBP-C 减慢肌球蛋白分离的作用，从而有效地加速了完整肌丝晶格中 β-心脏肌球蛋白的分离。长度扰动分析用于证明在完整的肌丝晶格中，β-心脏肌球蛋白的特征分离和募集速率被 Pi、cMyBP-C 的磷酸化和 cMyBP-C 的缺失所加速。结果表明，cMyBP-C 通常会减慢肌球蛋白的分离，包括 Pi 依赖性的分离，并且这种抑制在磷酸化或缺乏 cMyBP-C 时会被释放。
